Device and method for pressurized cryopreservation of a biological sample

ABSTRACT

A cryopreservation device ( 100 ) which is arranged for cryopreservation of a biological sample ( 1 ) comprises a pressure vessel ( 10 ) with a vessel wall ( 11 ) and an internal space ( 12 ) which is arranged to receive the biological sample ( 1 ), wherein the pressure vessel ( 10 ) is equipped with an actuating device ( 20 ) for cooling by lowering the temperature and raising the pressure in the pressure vessel ( 10 ) and configured for the cryopreservation of the biological sample ( 1 ). Said actuating device ( 20 ) is connected to the vessel wall ( 11 ) and comprises at least one pressure setting element ( 21 - 23, 31 ) and at least one of at least one cooling element ( 34 ) and at least one heat conducting element ( 35 - 38 ), wherein the actuating device ( 20 ) is configured for a time-dependent and/or location-dependent setting of the temperature and of the pressure in the pressure vessel ( 10 ). Methods are also described for cryopreservation of a biological sample ( 1 ), comprising biological cells ( 2 ) and a preservation medium ( 3 ), and methods for heating the biological sample ( 1 ) to maintain vitality.

The invention relates to a device for cryopreservation of a biological sample, comprising biological cells and an aqueous preservation medium, with a pressure vessel. The invention furthermore relates to a method for cryo-preservation of the biological sample and the use of a stabiliser substance with which a vitreous phase of water can be stabilised in the cryopreservation of biological samples. Furthermore, the invention refers to a method to heat the cryopreserved biological sample.

The low temperature preservation (cryopreservation) of cells is so far the only possibility of suspending vital processes reversibly at a cellular level (to maintain vitality) such that they revive after heating to physiological temperatures. Cryopreservation has no correspondence in nature. Whilst organisms, such as fish, are found embedded in ice in polar regions which maintain their vitality for a limited duration, these organisms are not completely frozen through but contain glycerine and other substances in their cells which serve to lower the freezing point, or they express proteins (so-called anti-freezing proteins) which influence the ice structure. Permanent preservation is not possible in this state because even at temperatures below 0° C., diffusion processes over weeks and months lead to the disintegration of the biological system. Permanent preservation would moreover require cooling, e.g. to a temperature of liquid nitrogen, at which the fluid in the cells would also freeze. In this case, however, the organisms can no longer be reanimated.

A special problem of cryopreservation is the formation of crystal ice (crystalline phase of water) inside and outside the cells which leads directly or indirectly to irreversible damage. Since the formation of ice is one of the primary reasons for cell damage, so-called cryoprotectants (anti-freeze additives, cryoadditives) have been sought for decades and added to the obligate physiological media.

Cryoprotectants typically comprise small molecules such as dimethyl sulfoxide (DMSO) which penetrate into the cells, or higher molecular substances such as sugar which remain in the medium outside the cells or on their surface. Cryoprotectants are frequently only effective in high, largely non-physiological concentrations (10% to 50%). Therefore they can only be added at lower than physiological temperatures (around 4° C.), must quickly penetrate into the cells and be washed out immediately after thawing.

Former developments in the cryopreservation of biological samples have been aimed at replacing cryoprotectants by physiologically and osmotically less problematical substances, reducing their concentration or foregoing them completely. With the exception of anti-freeze proteins of the organisms themselves, only few substance groups have been found since the discovery of the anti-freeze properties of DMSO and glycerine as well as a few sugars by Polge and Lovelock [1, 2, 3]. It has so far been assumed that the cryopreservation of cells is not possible without cell stress and cell repair processes with gene expressions etc. after thawing because the formation of the crystalline phase under physiological conditions cannot quite be avoided.

-   [1] Polge C, Smith A U, Parkes A S (1949), Nature 164: 666 -   [2] Lovelock J E (1954), Biochemical Journal 56(2):265-270 -   [3] Lovelock J E, Bishop M W H, 1959, Nature 183: 1394-1395

The success of cryopreservation can be characterised by a vitality rate (survival rate), e.g. by the quotients of the number of living cells after and before cryopreservation. It is known that the vitality rate depends on the type of cell, the volume and other different boundary conditions which have usually already been empirically optimised. In view of the necessity of a fast diffusion of the cryoprotectants into the cells and the regulated outward dissipation of heat, conventional cryopreservation methods have so far failed in microscopic objects such as tissues, organs and entire organisms without exception. Rather, conventional cryopreservation with the addition of cryoprotectants leads to practicable vitality rates only in suspended cells and very small tissue pieces (<0.5 mm³). It has not so far been possible to subject highly aqueous plant cells as well as a large number of animal cells, such as the oocytes of cats and other species, to cryopreservation at all. On the other hand, vitality rates in excess of 90% have been achieved with cryopreserved cancer cells.

The success of cryopreservation depends, in particular, on the physical-biological boundary conditions, e.g. on the properties of the water and those of the cryoprotectants. It has so far been assumed that cryoprotectants must penetrate into all cells by diffusion and that the heat must dissipate outwards in a short period (ms to min) because cooling can only take place from here. It is furthermore assumed that these conditions are satisfied only in very small objects (cell suspended in a nutrient solution with the addition of anti-freeze agents) due to the heat conductivity of the water which also dominates in the cells and due to the low diffusion speed of cryoprotectants through the cell membranes.

To avoid the creation of the crystalline phase, it has been attempted in practice to transfer biological samples to a vitreous or amorphous phase (vitrified state) through fast cooling. However, success has been limited even when using cryoprotectants. Up to a publication by J. L. M. Leunissen et al. [4] the general view was therefore that a vitrification of cellular cell suspensions could not be achieved without additives for physical reasons.

-   [4] J. L. M. Leunissen and H. Yi (2009) Journal of Microscopy, 235:     25-35

A method for cryomicroscopy is described by J. L. M. Leunissen et al. [4] which would lead vitrification to be expected: if a thin-walled copper tube with a diameter of <1 mm is filled with a cell suspension, closed gas-free at the ends by pressing them together and then frozen in a cooling fluid such as propane, nitrogen etc., very well maintained structures are found in deep temperature cryosections of the tubes which demonstrate vitrification. However, once the deep temperature cryosamples are heated, it becomes apparent that the cells did not survive the cooling process. The method described by J. L. M. Leunissen et al. [4] is not suitable for a heating of the sample such as to maintain vitality. It has therefore been assumed up to now that methods for cryomicroscopy do not permit revitalisation through reheating.

In view of the developing regenerative medicine and biotechnology and of environment protection and species preservation, there is an urgent interest in reducing or overcoming the disadvantages of conventional cryopreservation.

The objective of the invention is to provide an improved cryopreservation device using which the disadvantages of conventional techniques are overcome and which, in particular, permits a cryopreservation with a higher vitality rate and/or enlarged sample volumes. It is furthermore the objective of the invention to provide an improved method of cryopreservation in which the disadvantages of conventional techniques are overcome and which, in particular, permits the method conditions on cooling and/or thawing to be adjusted such that an improved vitality rate is achieved. Furthermore, it is an objective of the invention to provide an improved method to heat a cryopreserved biological sample with which the disadvantages of conventional techniques are overcome and which, in particular, permits any vitality-restricting influence of biological samples in the transition to a thawed state to be suppressed.

These objectives are solved by devices and methods with the features of the independent claims. Advantageous embodiments and applications of the invention are provided in the dependent claims.

In accordance with a first aspect of the invention, a cryopreservation device is provided which is adapted for the cryopreservation of a biological sample. The cryopreservation device comprises a pressure vessel with a vessel wall and an internal space which is arranged to receive the biological sample. The pressure vessel is configured to be cooled in a cooling bath of a cooling device up to a cryopreservation temperature, e.g. below −138° C. The pressure vessel is furthermore configured such that pressure can be applied to the internal space of the cooling vessel which is higher than the ambient atmospheric pressure. The pressure vessel is adapted for the permanent cryopreservation of the biological sample and storage at the cryopreservation temperature whilst maintaining the higher pressure in the pressure vessel.

In accordance with the invention, the pressure vessel has an actuating device which is suitable for a time and/or location-dependent setting of the temperature and of the pressure in the pressure vessel. The actuating device is connected to the vessel wall of the pressure vessel and is able to influence the setting of the cryopreservation temperature and of the pressure in the internal space of the pressure vessel depending on time and location. The actuating device is suitable to selectively control the cooling and/or heating of the biological sample in accordance with a predetermined temperature-time function. Furthermore, the actuating device is suitable to influence the temperature distribution in the pressure vessel. Furthermore, the actuating device is suitable to control the pressure in the pressure vessel in accordance with the predetermined pressure-time function. The temperature and pressure-time functions can be set relatively to each other. For example, the actuating device enables the biological sample to firstly be cooled and then a higher pressure applied to it, the cooling and increase in pressure to be conducted simultaneously or the pressure to firstly be increased and then the cryopreservation temperature set. Finally, the actuating device is suitable to control the place of pressure generation in the pressure vessel.

In accordance with the invention, the actuating device comprises at least one pressure setting element, at least one cooling element and/or at least one heat conducting element. The pressure setting element is configured such as to set the pressure-time function, a location of a primary pressure input and/or the amount of the pressure which is applied to the internal space of the pressure vessel. Accordingly, the cooling element and/or the heat conducting element, possibly in connection with the effect of the cooling device, are configured so as to control the temperature-time function, the spatial temperature distribution and the cryopreservation temperature.

The biological sample contains biological cells and an aqueous preservation medium. The biological cells comprise individual cells, such as individual stem cells, precursor cells, fibroblasts, gametes, groups of cells, in particular from the named cell types, pieces of tissue or organs or their parts. The biological cells can even form complete organisms, in particular small organisms, such as worms or insects. The preservation medium comprises an aqueous physiological medium (culture medium, cultivation medium) as known in the cultivation of biological cells.

In accordance with a second aspect of the invention, a method for the cryopreservation of the biological sample is provided in which the biological sample is arranged in a pressure vessel and the pressure vessel is cooled down in a cooling device until the biological sample in the cryopreserved state has been transferred to an at least partially vitreous phase. In accordance with the invention, a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel takes place using an actuating device with at least one pressure setting element, at least one cooling element and/or at least one heat conducting element. The cryopreservation device in accordance with the first aspect of the invention is used by preference to conduct the inventive method of cryopreservation of biological samples.

In accordance with a third aspect of the invention, a method is provided to heat a biological sample, comprising biological cells and a preservation medium in a frozen state which is located in a vitreous phase in a pressure vessel. According to the invention, the temperature of the pressure vessel and of the biological sample is increased and during the temperature increase in the pressure vessel a higher pressure is maintained above the ambient atmospheric pressure. The method is preferably executed to heat the biological sample using the cryopreservation device in accordance with the first aspect of the invention.

In accordance with a fourth aspect of the invention, it is proposed to use at least one stabiliser substance (i.e. an individual substance or a mixture of several substances) in the cryopreservation of biological samples as a component of the preservation medium which is suitable to maintain a vitreous state of the supercooled melt without crystallisation preferably up to the transition to the liquid state whilst increasing the temperature of the biological sample comprising biological cells and a preservation medium.

The invention is based on the recognition that on freezing aqueous systems the formation of the crystalline phase is avoided and instead the vitreous phase (amorphous phase) can be generated by applying pressure to the aqueous system. The transition of the biological sample to the cryopreserved state, the cryopreservation and/or the heating of the cryopreserved samples are conducted in an area of the phase diagram of the aqueous system in which preferably the vitreous phase is formed. Since the survival rate of the biological cells in the biological sample in the vitreous phase is considerably higher than in the crystalline phase, the invention facilitates a higher vitality rate of the cryo-preservation.

In conventional cryomicroscopy using pressure-tight sample tubes (see above, [4]) an increased pressure was already generated in the closed sample tubes. However, with the conventional technique there is no degree of freedom to influence the pressure or temperature characteristic during freezing, during cryopreservation or during selective heating in accordance with the set time function. The inventors have determined that ice avoided during rapid cooling is inevitably created—particularly during thawing—with all its negative effects on the sample, thereby destroying the cells. The inventive provision of the actuating device advantageously permits the pressure-temperature processes to be controlled in terms of time and/or space such that the vitreous phase of the biological sample is primarily generated and maintained in the internal space of the pressure vessel. Contrary to the conventional technique, the actuating device permits the pressure and temperature parameters to be varied selectively during freezing and/or thawing in order to achieve a maximum vitality rate. This advantageously provides a real cryopreservation with the possibility of rethawing and revitalisation of the cells in the biological sample whilst the technique of J. M. L. Leunissen et al. [4] merely represents cryopreparation for microscopic examinations.

In accordance with the above-mentioned third aspect of the invention, it is suggested in particular to maintain the vitreous state of the sample, in particular of the preservation medium, maintaining pressure until the water contained in the sample melts. This is advantageously facilitated by thawing cryopreserved biological samples, as those described by J. M. L. Leunissen et al. [4], with an extremely good maintenance of structure and vitrified cell suspensions such that living cells exist. This has not so far been successful in any freeze-pressure shock method as used for cryomicroscopy. In order to achieve the above-mentioned stabilisation, preferably one stabiliser substance is added to the preservation medium which is suitable to stabilise the vitreous phase of the supercooled melt on increasing the temperature of the biological sample preferably up to the melt. The inventive cryopreservation device advantageously facilitates the influencing of the path of the sample conditions through the phase diagram past a solid/fluid phase transition.

In accordance with a preferred embodiment of the invention, the actuating device comprises at least one pressure setting element which is connected to the vessel wall and/or the internal space of the pressure vessel. Advantageously, different variants of the pressure setting element are possible which, depending on the preservation task and the spatial conditions of the cryopreservation, can be selected individually or in combination. In accordance with a first variant, at least one pressure screw can be provided in the vessel wall of the pressure vessel. The vessel wall contains a threaded opening for the fluid-tight reception of the pressure screw. Screwing the pressure screw into the threaded opening serves to reduce the free volume of the internal space in the pressure vessel and to permit the pressure in the pressure vessel to be increased accordingly. According to a second variant, an expansion area can be provided which is connected with the internal space of the pressure vessel and is adapted to receive a fluid or gaseous expansion medium. When the expansion medium expands in the expansion area the free volume in the internal space is reduced accordingly and the pressure in the pressure vessel increased. In accordance with a third variant, the pressure setting element can comprise a pressure clamp which acts on the vessel wall from outside. In this case, the vessel wall is formed of a flexible material in order to transfer the mechanical pressure exercised by the pressure clamp to the internal space of the pressure vessel. Advantageously, the pressure clamp can be adapted with cooling openings in order to accelerate the cooling of the pressure vessel in the cooling bath of the cooling device. Furthermore, the pressure clamp can be designed for a mechanical or electrical actuation.

According to a specially preferred embodiment of the invention, the expansion area comprises at least one hollow duct to accommodate the expansion medium which communicates with the internal space of the pressure vessel and which protrudes from the pressure vessel. The duct contains water, for example, an aqueous solution or parts of the biological sample. The protrusion of the duct from the pressure vessel causes the expansion medium in the duct to be spatially separated from the internal space of the pressure vessel. This advantageously facilitates a cooling of the duct in the cooling bath of the cooling device whilst the remaining pressure vessel is still at a higher temperature, e.g. at room temperature. Crystalline ice can be generated in the duct which extends through to the internal space, decreasing the remaining volume and therefore causing an increase in the pressure in the internal space. In accordance with a preferred variant, the duct has branches. Advantageously, this facilitates an enlargement of the volume of the expansion area compared with an individual duct without branches. At the same time the branched duct permits the time function of the pressure generation in the pressure vessel to be influenced. Alternatively and additionally, several ducts can be provided which protrude from the pressure vessel in different directions. Advantageously, this permits the pressure generation to be influenced, depending on the alignment of the pressure vessel relative to the cooling bath of the cooling device.

If the actuating device provided by the invention comprises a cooling element, this is preferably formed by a cooling line which is arranged in the pressure vessel. The cooling line runs through the internal space of the vessel. It is designed to have a cooling agent, such as liquid nitrogen, run through it. Advantageously, the cooling line facilitates a setting of the start and of the time function of the temperature reduction in the pressure vessel.

If the actuating device provided by the invention comprises a heat conducting element, this is preferably formed by an outside profile on the outer side of the pressure vessel, an inside profile on the inner side of the pressure vessel and/or heat conducting bodies in the inside of the pressure vessel. These variants of heat conducting elements permit the heat transport from the pressure vessel to the cooling bath to be accelerated during the cooling of the pressure vessel.

A further advantage of the inventive cryopreservation device is that the pressure vessel can be manufactured in a large number of geometric shapes. Variants of the invention are given particular preference in which the vessel wall of the pressure vessel has the shape of a tube, a sphere or a cylinder, e.g. of a flat cylinder (box). A tubular pressure vessel can be straight or curved. In both cases the spatial distribution of the temperature and pressure setting in the pressure vessel can be influenced by its shape.

Further advantageous features of the inventive cryopreservation device refer to the shape of the internal space of the pressure vessel. According to a variant of the invention, an inner vessel may be provided in the internal space which is arranged to receive the biological sample. In this case, the biological sample in the inner vessel is separated in terms of the substance from the remaining volume of the internal space. This facilitates the influencing of the vitreous phase in the direct vicinity of the biological sample. In accordance with a further variant, a substrate can be arranged in the internal space which is suitable to adherent receive biological cells which are part of the biological sample. The substrate can, for example, be arranged in the inner vessel. Substrate materials are suitable as substrates which are used for adherent cell cultures such as plastic or glass. In accordance with a further variant, a segmentation of the internal space can be provided in internal space sections. Advantageously, the segmentation facilitates the selective setting of different pressure-temperature conditions in each of the internal space sections. In accordance with a further variant, a sensor device can be arranged in the internal space which comprises at least one temperature sensor and/or at least one pressure sensor. Advantageously, the sensor device facilitates a measurement of the temperature and/or of the pressure in the internal space. The cryopreservation can be controlled depending on the at least one signal of the sensor device. Finally, in a further variant of the invention a substance reservoir can be arranged in the internal space which is suitable to release a substance into the internal space. The substance reservoir comprises, for example, hollow spheres which may be destroyed under the effect of a higher pressure in the internal space in order to release a substance. The variants specified for the internal space design can be provided individually or in combination.

In accordance with a further advantageous embodiment of the invention, the vessel wall can be provided with an optical unit. The optical unit comprises an imaging optic which is set up for a visual observation of the internal space of the pressure vessel. The optical unit facilitates a visual monitoring of the state of the biological sample during cryopreservation.

Advantageously, different variants of the pressure and temperature setting exist in the pressure vessel in order to achieve the required cryopreservation conditions in different ways in the pressure-temperature phase diagram of the biological sample. For example, in accordance with the first variant it is possible to first increase the pressure in the pressure vessel with the at least one pressure setting element and then to reduce the pressure in the pressure vessel with the cooling device. The pressure increase and the temperature reduction are conducted in accordance with predetermined separate time functions. In accordance with the second variant, the increase in the temperature and the reduction in temperature can be set such that the respective time functions overlap by starting the lowering of the temperature before achieving the final pressure or vice versa by starting the increase in pressure before achieving the cryopreservation temperature. Finally, in accordance with a further variant it is possible to firstly reduce the temperature in the pressure vessel up to the cryopreservation temperature and then to increase the pressure in the pressure vessel. Which of the variants stated is selected will depend on the conditions of the specific cryopreservation task, in particular on the design of the cryopreservation device and the composition of the biological sample. The ideal variant can be selected empirically by tests in which the vitality rate of the biological sample is tested for the different variants under specific conditions of application. It is possible in the same way to select the pressure and temperature time function, particularly with respect to the speed of pressure increase and temperature reduction and/or the shape of the function, such as a stepped shape.

The pressure-temperature setting is simplified if the pressure setting element in accordance with a preferred embodiment of the invention comprises an expansion area in the form of a hollow duct protruding from the pressure vessel. The pressure can be increased in the pressure vessel by immersing the at least one duct in the cooling bath of the cooling device. An expansion medium in the at least one duct expands so that the pressure in the remaining pressure vessel increases. Finally, the remaining pressure vessel is immersed in the cooling bath of the cooling device in order to set the cryopreservation temperature for the biological sample in the internal space of the pressure vessel.

Preferably, a permanent storage of the biological sample is provided whilst maintaining the increased pressure, in particular in liquid nitrogen or in the vapour of the liquid nitrogen. The sample is stored by special preference in the pressure vessel.

In accordance with a specially preferred embodiment of the cryopreservation method, the preservation medium contains at least one stabiliser substance which is suitable to stabilise (maintain) the vitreous phase of the supercooled melt preferably up to the transition to the fluid state whilst increasing the temperature of the biological sample.

Advantageously, the stabiliser substance brings about a situation in which the vitreous phase remains up to higher temperatures than would be the case without the stabiliser substance. The vitreous phase is maintained for longer during thawing and the formation of the crystalline phase is avoided. Furthermore, the glass transition temperature of the preservation medium is increased by the stabiliser substance. The stabiliser substance produces a lower number of nucleation sources in the preservation medium so that the nucleation probability is reduced and the formation of ice during thawing minimised.

In accordance with this embodiment, the above objectives are solved by at least one stabiliser substance being added to the biological sample, e.g. a cell suspension. The at least one stabiliser substance is used as was used only under certain conditions or not at all for the conventional cryopreservation or, in accordance with the invention, develops its effect at far lower concentrations and this is a different effect than that of the known cryoprotectants. Substances are preferably selected as stabiliser substance which do not penetrate the cells at normal pressure and which therefore are not normally used in conventional cryopreservation of cells and tissues. The noteworthy aspect of using the stabiliser substance is that experiments of the inventors have shown that with an addition of Percoll or Ficoll, for example, in the range of a few percent mammalian cells survived the pressure shock freezing and thawing procedure analogue to publication [4] with a high survival rate (>97%). This is all the more surprising in view of the fact that a penetration into the cells is not to be assumed.

The surprising effect of the stabiliser substance is that for the first time during the thawing of the sample it permits the return path via the combination of high pressurefast deep cooling and pressure-controlled heating virtually without influencing the vitality of the cells. It is therefore the combination of at least one substance dissolved in the preservation medium which permits the return from the vitrified phase by influencing the water structure and the existence of nucleation sources.

The stabiliser substance differs in terms of substance, in terms of its effect and/or with relation to the preferred selected concentration of conventional cryopreservation. Unlike the stabiliser substance, conventional cryoprotectants selectively increase the number of nucleation sources in order to promote the formation of a large as possible number of ice crystals during freezing. However, as the number of ice crystals increase, their chance of growing in size reduces so that large crystals are prevented by conventional cryoprotectants. A distribution and high motility in the preservation medium through to the cells is required in conventional cryoprotectants.

The stabiliser substance is preferably selected from at least one of the substance groups which comprise long-chain uncharged polymers with a molecular weight greater than 500 g/mol, in particular greater than 1000 g/mol, monosaccharides, di- and oligosaccharides, polysaccharides, starch derivatives such as starch hydrolysis products, sugar alcohols, water-soluble polymers, colloids (nanoparticle dispersions), in particular with silver, gold, diamond and/or nanotube particles, dendrimers, polycations and polyanions. Long-chain polysaccharides proved to be particularly suitable, in particular hydrophilic copolymerisates made from saccharose and epichlorohydrin (Ficoll, reg. name), and/or polyvinylpyrrolidone, in particular silica gel coated with polyvinylpyrrolidone (Percoll, reg. name) with a molecular mass of between 2,000 and several million g/mol. Nanoparticle dispersions, in particular silver, gold, diamond and/or nanotube particles are particularly advantageous because they are suitable to reduce the heat conductivity of the preservation medium. Advantageously, the stabiliser substance is biocompatible so that the cells in the biological sample are not unfavourably influenced by the stabiliser substance.

The concentration (%=vol. %) of the stabiliser substance is preferably smaller than 30%, in particular preferably less than 20%, in particular smaller than 10%, such as for example smaller than 3% or smaller than 1%. A preferred minimum concentration is 0.1%.

In accordance with a further preferred embodiment of the invention, the stabiliser substance in the preservation medium is positioned outside the biological cells. The cells remain free from the stabiliser substance which has advantages for the vitality after thawing of the sample.

A further advantage of the stabiliser substance can be that it alters its properties, in particular structure and/or molecular weight, under the impact of the increased pressure and the reduced temperature during cryopreservation. For example, molecules of the stabiliser substance can be fragmented so that they can diffuse into the inside of the cells in order to achieve an additional cryoprotective effect here.

It is emphasised that with the addition of the stabiliser substance to the preservation medium it is not necessary for an increased pressure to be maintained in the pressure vessel until the biological sample has achieved the transition from the supercooled melt to the liquid state. In this case, the pressure can drop even before achieving the transition, in particular down to atmospheric pressure.

According to a preferred variant of the above-mentioned third aspect of the invention, a method is thereby provided to heat a biological sample, comprising biological cells and a preservation medium in a vitrified state, which with a vitreous phase is located in a pressure vessel, wherein the temperature of the pressure vessel and of the biological sample is increased until the biological sample reaches the liquid state and wherein the preservation medium contains the at least one stabiliser substance which is suitable to stabilise the vitreous state on increasing the temperature of the biological probe preferably up to the transition to the liquid state.

In accordance with a preferred embodiment of the invention, the pressure in the pressure vessel is reduced on reaching the transition from vitreous state of the supercooled melt through to liquid state. Damage to the biological sample after reaching the liquid state is minimised here. Special preference is given to the reduction of the pressure in the pressure vessel instantaneously, i.e. in particular in steps and with negligible delay.

The pressure reduction can be achieved by releasing the pressure vessel, e.g. opening the pressure vessel such that a pressure balance with the outer atmospheric pressure is achieved. Advantageously, a release of the pressure vessel is not absolutely necessary, however. Rather, the pressure reduction can also be achieved by a contraction of the biological sample at the transition from the supercooled melt to the liquid state. The volume of the sample can reduce in accordance with the processes explained with reference to the FIGS. 1 to 4 so that the pressure in the pressure vessel drops.

In accordance with a further preferred embodiment of the invention, the increased pressure above atmospheric pressure is at least 100 MPa, in particular at least 150 MPa and/or at the most 300 MPa, in particular 250 MPa at the most. These pressure areas have proven to be particularly advantageous for a fast transition from the vitreous to the liquid state and vice versa.

Further details and advantages of the invention are described in the following, making reference to the attached drawings, which show in:

FIGS. 1 to 4: phase diagrams with illustrations of different phases of water; and

FIG. 5: embodiments of the inventive cryopreservation device;

FIG. 6: graphic representations of different variants of the pressure and temperature-time functions;

FIGS. 7 to 11: experimental results which illustrate the effect of a cryoprotectant or of a stabiliser substance;

FIG. 12: diagrammatic sections of different variants of a pressure vessel which is provided with heat conducting and/or cooling elements;

FIGS. 13 to 19: further embodiments of inventive cryopreservation devices and their use;

FIGS. 20 to 28: further embodiments of inventive cryopreservation devices in which the pressure vessel has the shape of a flat cylinder;

FIG. 29: a diagrammatic illustration of an optical unit in the vessel wall of a pressure vessel;

FIG. 30: a further embodiment of the inventive cryopreservation device and its use;

FIGS. 31 to 35: further embodiments of inventive cryopreservation devices in which the pressure vessel has the shape of a sphere or of a cylinder;

FIG. 36: a further embodiment of the inventive cryopreservation device;

FIGS. 37 to 39: further embodiments of inventive cryopreservation devices in which a substrate is arranged in the internal space of the pressure vessel;

FIGS. 40 and 41: further embodiments of inventive cryopreservation devices with a segmentation for the internal space of the pressure vessel; and

FIG. 42: a further embodiment of the inventive cryopreservation device and its use.

The invention will firstly be described in the following by explanation of findings of the inventors and then by specifying details of the cryopreservation device and the method. It is emphasised that the following theoretical considerations serve as an approach to explain the outstanding vitality rates achieved with the inventive cryopreservation. However, the implementation of the invention is not bound by the completeness and correctness of the theoretical considerations.

Theoretical Considerations of the Phase Diagrams of Aqueous Systems and the Effect of Stabiliser Substances

Cryopreservation has so far been described empirically and using simplified assumptions. The empirical approach results from the complexity of the composition of the cytoplasm of the biological cells. Since the cytoplasm contains hundreds of proteins, nucleotides and a large number of carbohydrates, ions of numerous elements, nano-scale systems such as membranes, organelles and structure elements such as cytoskeleton and water bonded to the surfaces, phase diagrams of water can be used to only a restricted extent. Nevertheless, reference is made to phase diagrams of water as shown in FIGS. 1, 2 and 4 to illustrate the explanation of the findings of the inventors.

The pressure-temperature phase diagram of water (FIG. 1, source: Jackson et al., J. Phys. Chem. (1997), cited in www.wikipedia.de, keyword water) shows that so-called Ih (hexagonal ice) is formed on cooling under normal pressure (0.1 MPa=0.0001 GPa, line on the ordinate axis). This assumes a greater volume compared to the liquid phase (approx. 110) leading to mechanical tensions (pressure) in delimited volumes. During formation of ice, multi-component solutions (ions and other molecular constituents are concentrated) decombine as would not otherwise occur in the cytoplasm and this should therefore be avoided.

As the phase diagram in FIG. 1 shows, further ice structures exist which are known not to exhibit the specified volume increase at higher pressure. However, biological objects in unfrozen state would be destroyed under these pressures (e.g. >0.1 GPa).

In extended phase diagrams of water and of its metastable states, FIG. 2 shows that possibilities exist to influence the formation of ice. By adding substances, the freezing point can, for example, be lowered in a stable manner (colligative effects). This is the principle used by organisms in nature. They then freeze only at −10° C. or −20° C. for example. Furthermore, an aqueous solution can be supercooled under certain conditions. Ice melts with pure water and at normal pressure at 0° C. but it does not freeze at this temperature. Nucleation is necessary for this. In a so-called homogenous nucleation, freezing is a stochastic process which is triggered by the smallest disturbances. In both cases, the range of the liquid phase is extended down to below −50° C. to −70° C. Under consideration of the pressure range up to 300 MPa=3,000 atm (FIG. 2, right hand side) the following results:

-   -   1. Up to 200 MPa so-called LDL (Low Density Liquid, supercooled         liquid) water exists in its aggregate states with all the         anomalies occurring at normal pressure.     -   2. In addition HDL (High Density Liquid) states arise which may         possibly be more favourable for cryopreservation. However, these         are unphysiologically high pressures (in the deep sea a maximum         of 100 MPa is achieved).

In metastable states a limit temperature below that which a vitreous state of water could be assumed even with physiological pressures is to be found at temperatures of around 136 K=−137° C. Water then freezes like glass, namely amorphously, i.e. without the formation of crystals. The water molecules then remain where they were. This is a metastable state, the desirable state which is aspired to as found when deep-freezing biological objects. It is called “vitrification”. The vitrification requires a very high cooling rate (>10⁶°/s) with the rapid fluctuation of the water molecules which, given the dimension of a cell (>10 μm), cannot be reached due to the neighbouring heat conductivity of the water (max. a few 10⁴°/s). With increasing size of the objects to be frozen, the cooling rate interval becomes increasingly dramatic (many powers of ten) so that a true vitrification (formation of a vitreous phase without additives) has not so far been possible.

The inventors have determined that in view of the anomaly of water in the LDL range the freezing point can be lowered by increasing pressure. In the transition from LDL to HDL this process is exactly reversed so that a pressure of around 200 MPa is the preferred pressure upper limit which is still effective to lower the freezing point. Thereafter the freezing point increases as is normal in any other liquid as the pressure increases.

The path to vitrification (vitreous phase) is the shortest at this pressure (see FIG. 2, right hand side diagram, vertical line in the middle at 0.2 GPa); only a little more than 100 degrees must be passed through rapidly, which is why shock freezing is used. A long-standing paradox of cryopreservation is that the structures of the cells are best maintained with pressure-shock freezing but after thawing no cells survive. On the other hand, it is known that survival rates of over 90% can be achieved with slow cooling with even microscopically visible ice domain formation and damage of the cells (membrane leaks). The inventors have determined that the reason for this is primarily to be found in the thawing process. The critical range from −137° C. to melting point (perhaps −5 to −15° C.) is run through once again so that the avoided or reduced Ih ice formation during freezing does occur. At low pressure even HD ice quickly changes into the LD ice form. Added to this is that in accordance with the thermal impulses (k*T) starting from −100° C. with rising temperature the probability of a water molecule changing place is relevant also in the frozen phase. This leads to the so-called “migratory growth” of large ice crystals because they gain size at the expense of the smaller ones (due to the surface tension differences). Therefore, long-term storage must be conducted below the glass transition temperature (−137° C.).

However, there is another way of achieving vitrification or at least a “pseudo vitrification” by adding additives which lead to very small ice domains. This is not a truly physiological way but it has been used for freezing for a very long time and also functions at normal pressure.

The possibility exists in principle to increase the glass transition temperature through additives. Adding Trehalose permits the glass transition temperature, for example, to be shifted to almost 0° C., which would be ideal for biological objects. The requisite concentrations of Trehalose are, however, above any physiological compatibility for living cells. The gains achieved by lowering the freezing point are small (FIG. 3, lowering the freezing point and shifting the glass transition temperature by adding Trehalose) and this applies to virtually all similar substance additives.

FIG. 4 shows once again that in the LD range it is necessary to pass through a critical range described as “no man's land” in the diagrams during cooling and thawing. However, the solution to the problem would be precisely to reach this area or to aspire to complete vitrification.

In accordance with a practical example, it is intended to prepare a biological sample in a known manner with biological cells and a preservation medium at room temperature and under atmospheric pressure. At least one stabiliser substance is added to the preservation medium, preferably with a concentration smaller than or equal to 5% or this is done during the cooling process or in the deep-frozen state or before thawing. Higher concentrations can also be used, however.

Suitable substance groups for the stabiliser substance, in particular for use with the embodiments of the cryopreservation device and the methods to cool or heat biological samples described below are as follows:

Substance class Substance examples Alcohols Ethyl glycol Monosaccharides Glucose Fructose Mannose Galactose Ribose Xylose Arabinose as well as non-naturally occurring sugars Di- and oligo-saccharides Sacchrarose (sugar cane) Lactose Maltose Trehalose Cellobiose Polyether Polyethylene glycol Molecular mass 1000 to 35,000 Polysaccharides Cellulose Hemicellulose Amylose Glycogen Pectins Dextran (artificial) Alginates Ficoll, in particular with a molecular weight of 2500 g/mol to 2.5 million g/mol

Further media with which excellent experimental results have been achieved using the embodiments of the cryopreservation device described below and methods to cool and heat biological samples are as follows:

Cryomedium according Cryomedium according Dextran to Mazur to Matsumura 30% dextran Ethylene glycol 10% Ethylene glycol 20% (3.23M) (6.5M) Acetamide 10.7% e-poly-L-Lysine 10% (3.27M) Ficoll 70 24% (3.5 mM) Sucrose 10.9% Sucrose 25.7% (0.4M) (0.75M) BSA & glucose (low concentrations) 325 Mosm 3.4 Osm 6.7 Osm

In the experiments of the inventors the cryomedium according to Mazur with an addition of ethylene glycol and dextran proved to be advantageous.

Embodiments of the Cryopreservation Device and the Methods to Cool or Heat Biological Samples

FIGS. 5A to 5C show embodiments of the inventive cryopreservation device 100 which has a pressure vessel 10 and an actuating device 20. The pressure device 10 has a vessel wall 11 in the shape of a tube (small tube) the inside of which forms the internal space 12 of the pressure vessel 10. The actuating device 20 comprises pressure setting elements which are each created by pressure screws 21 at the axial ends of the pressure vessel 10. A third pressure screw 21 can be provided protruding radially at the vessel wall 11, for example along the half axial length of the pressure vessel 10 (FIG. 5B).

The pressure vessel 10 preferably has an outer diameter which is smaller than or equal to 5 mm and by special preference smaller than or equal to 2 mm or 1 mm, e.g. 0.5 mm. The axial length of the pressure vessel 10 has been selected, for example, in the range of 10 mm to 20 cm. The thickness of the pressure wall 11 is, for example, ¼ to 1/10 of the outer diameter of the pressure vessel. The pressure wall 11 is made, for example, from stainless steel, aluminium, gold or silver. Alternatively, further metals or alloys can be used which have a high heat conductivity for fast cooling in the internal space 12 and a pressure resistance for pressures of up to 100 MPa, for example. Furthermore, the pressure vessel can be made of a plastic or a composite material, e.g. plastic-metal composite.

The pressure vessel 10 has internal threads at its axial ends to receive the pressure screws 21. The vessel wall 11 has a threaded piece to accommodate the radially protruding pressure screw 21 (FIG. 5B). The pressure screws 21 can be used additionally as bleed screw. Unlike the illustrations in FIG. 5, a single pressure screw 21 can be provided as actuating device (see for example FIG. 14A).

In accordance with FIG. 5C, the internal space 12 of the pressure vessel 10 can be subdivided into individual internal space segments 15. The segmentation with internal space segments 15 has proven to be advantageous for the vitality rate of biological cells which is a maximum in the middle internal space segments 15.

For the cryopreservation of a biological sample 1 comprising, for example, biological cells 2 in a preservation medium 3, the sample 1 is filled into the internal space 12 of the pressure vessel 10 (see partly cross-sectioned view of the vessel wall 11 in FIG. 5A). The preservation medium contains one or several stabiliser substances such as dextran with a concentration of 30% mixed with 10% ethylene glycol. If the inner space 12 is completely filled, the pressure screws 21 are closed such that the internal space 12 is free from bubbles. In order to fill the internal space free from bubbles, the inner side of the vessel wall 11 must be completely wetted by the biological sample 1. For this purpose, the inner side of the vessel wall 11 can be coated in a hydrophilic manner. Furthermore, one of the pressure screws 21 can be used to bleed the system. As a result of penetration of the pressure screws 21 an increased pressure can be set in the internal space 11 of the pressure vessel 10, e.g. in the range from 0.1 MPa to 300 MPa even at room temperature. The amount of pressure can be determined by calibration tests or using a sensor device 16 (see below). As an option, the segmentation into internal space segments 15 (FIG. 5C) can be provided in or after this phase by squashing the vessel wall together.

For the cryopreservation of the biological samples, the filled cryopreservation device 100 is cooled in the cooling bath of a cooling device (not shown in FIG. 5, see FIG. 13, for example). For this purpose, the pressure vessel is preferably immersed horizontally into the cooling bath so that the cooling essentially takes place simultaneously along the entire axial length of the pressure vessel 10. The cooling bath comprises, for example, liquid propane, liquid nitrogen or another liquid gas.

On immersion in the cooling bath ice firstly forms on the inner side of the vessel wall 11. Since the crystalline phase is firstly formed, an expansion takes place which leads to a pressure increase in the internal space up to a final pressure of 200 MPa. Above this pressure further ice growth is ruled out so that the remainder of the biological sample 1 moves to a vitreous (vitrified) state or at least does not exhibit a hexagonally crystalline phase.

Once the cryopreservation device 100 in the cooling bath has reached the cryopreservation temperature, e.g. −197° C., the further cryopreservation can take place in the cooling bath or in storage vessel (not shown) which has been cooled to a temperature of, for example, −140° C. through liquid nitrogen or through vapour of the liquid nitrogen.

For the heating of the biological sample 1 so as to maintain vitality, the cryopreservation device 100 is immersed in a heating bath (liquid bath with a temperature above 0° C.) comprising, for example, water, alcohol or an oil. Immersion is similarly conducted horizontally by preference and at a high speed so that the increased pressure in the pressure vessel 10 is maintained until the crystalline ice which is formed melts. Finally, the pressure drops suddenly to 0.1 MPa, for example.

FIGS. 6A to 6D show four typical temperature and pressure-time functions as may be realised in cryopreservation (left part of the curves) or during thawing (right part of the curves) with the cryopreservation device 100 in accordance with the embodiments of the invention explained here. FIG. 6A shows, for example, how during freezing the pressure is initially increased. For this purpose, pressure screws 21 as shown in FIG. 5 are used. Only after the pressure of 200 MPa is reached is the temperature reduced by immersion (dipping) of the cryopreservation device 100 into the cooling bath. During thawing pressure can be released and then the biological sample heated to room temperature. FIG. 6B shows the opposite variant in which during cryopreservation the temperature is firstly reduced and then the pressure increased. In this case the pressure screws 21 are actuated only after reaching the cryopreservation temperature of, for example, −200° C. During thawing, pressure can be released as the temperature rises. FIG. 6C shows a more complicated temperature-time function which can be selected as dependent on the specific preservation conditions. Using the inventive cryopreservation device 100 a pressure temperature curve can also be realised as would exist with a cryopreservation device without actuating device (FIG. 6D), whereby the pressure build-up is started directly after falling below the freezing point by the creation of the crystalline phase of ice in the biological sample. The shape of the time functions is influenced by the actuating device used in the invention. In accordance with a further variant of a temperature and pressure-time function (not shown) the increased pressure can be set throughout the entire preservation time.

FIG. 7 shows a sequence of microscopic images of a section of sample 1 with cells 2 at room temperature after a cryoprotectant has been added to the preservation medium 3. The time (in seconds, s) shows the dependence on time after adding the stabiliser substance to sample 1. It becomes clear that a considerable shrinking process takes place in a short period of time. The inventors have found that this shrinking can be advantageous for the achievement of high vitality rates.

FIGS. 8 and 9 show the dramatic shrinking of the cells (here HeLa cells) virtually to the osmotic residual volume which is advantageous for high vitality rates. The round form of the cells (FIGS. 9A to 9C, top left) is lost completely (FIGS. 9A to 9C, right and bottom). The reduction in the diameter of the cells was determined with an invitrogenic measurement system (diagram in FIG. 8), but does not completely reflect the osmotic shrinking because the part of deviation from the spherical form is not considered. The cells consequently shrink more greatly than shown by the diagram figures.

FIG. 10 shows the shrunken cells in an electron-microscopic section. The extremely strong shrinking can be recognised which is achieved in the cryomedium with one of the above-mentioned compositions so that the cell membrane system is greatly folded. FIG. 11 shows the structural changes of adherent cells (HeLa). Here, too, the osmotic shrinking occurs very quickly (after seconds).

Fluorescence-microscope investigations with a different presentation of the cell nucleus and of the Golgi apparatus (not shown here) have shown that the shrinking merely compacts the cytoplasma components but does not influence the cell nuclei and other important cell organelles; this is of great advantage to freezing and thawing such as to maintain vitality.

FIG. 12 shows variants of the inventive heat conducting and/or cooling elements intended. For purposes of comparison, FIG. 12A firstly shows the circular cross-section of the tubular pressure vessel 10 (see FIG. 5). FIGS. 12B to 12I show embodiments of the inventive cryopreservation device as diagrammatic sectional views of the pressure vessel 10 in which the actuating device is suitable for a setting of the temperature using at least one heat conducting element or at least one cooling element. In accordance with FIG. 12B, heat conducting elements comprise outer profiles 35 which are arranged radially protruding on an outer side of the vessel wall 11. The outer profiles 35 accelerate the cooling or heating of the pressure vessel 10 on immersion in the cooling bath or the warm liquid bath. In accordance with FIG. 12C, the inner profiles 36 are arranged on an inner side of the vessel wall 11. The inner profiles 36 similarly support the heat transport from or to the biological sample in the pressure vessel 10.

FIGS. 12D to 12G illustrate the pressure vessel 10 with a hexagonal cross-section. The outer side of the vessel wall 11 forms the outer profile 35 which provides a large surface for the wetting with a cooling agent or thawing agent and is therefore suitable to influence the temperature-time function during cooling or thawing. In accordance with FIGS. 12E, 12F and 12G, heat conducting bodies 37 in the shape of partition walls (FIG. 12E), inserted filaments or spheres (FIG. 12F) or colloidal particles (FIG. 12G) are provided additionally in the internal space of the pressure vessel 10. The colloidal particles 37 in accordance with FIG. 12G are shown diagrammatically enlarged but in practice can have dimensions in the sub-micrometre range. The cooling or thawing speeds can be increased advantageously with the outer profiles 35, inner profiles 36 and/or heat conducting bodies 37. The heat conducting elements are preferably made from silver or gold or other substances with high heat conductivity. In accordance with further variants, the heat conductivity between the internal space 12 and the outer environment of the pressure vessel 10 can be influenced by a coating of the vessel wall 11 on its inner or outer side, e.g. with diamond or other substances with high heat conductivity.

FIG. 12H shows a variant of the invention in which a cooling line 34 runs through the inner space 12 of the pressure vessel 10 as cooling element. The cooling line 34 is connected with a cooling medium reservoir, and it is adapted in order to be supplied with a cooling medium such as liquid nitrogen or a cooling gas. Alternatively, a cooling line 34 can run next to the internal space 12 as shown diagrammatically in FIG. 12I.

FIG. 13 shows a further embodiment of the inventive cryopreservation device 100 (FIG. 13A) and its use in cooling (FIG. 13B) and thawing (FIG. 13C) of the biological sample with cells 2 in the preservation medium 3. In this embodiment of the invention the actuating device comprises three pressure setting elements comprising pressure screws 21 and an expansion area 22 for the time and/or location-dependent setting of the temperature and of the pressure in the pressure vessel 10. The pressure screws 21 are provided for the generation of pressure and/or to bleed the internal space 12 of the pressure vessel 10 (see FIG. 5). The expansion area 22 comprises a duct which protrudes radially from the pressure vessel 10 which at one end is connected to the internal space 12 of the pressure vessel 10 via an opening in the vessel wall 12 and whose opposite free end is closed. A filter 29 can be provided between the internal space 12 and the expansion area 22 in order to prevent the penetration of biological cells into the expansion area 22.

The expansion area 22 is made, for example, from the same material as the vessel wall 12 of the pressure vessel 10. The expansion area 22 is adapted to receive a liquid expansion medium that expands during cooling. The expansion medium comprises, for example, the aqueous preservation medium of the biological sample or alternatively a different aqueous liquid which is suitable to form the crystalline phase of water. The dimensions (length, inner diameter, outer diameter) of the expansion area 22 can be selected by the user depending on the specific preservation conditions.

The expansion area 22 permits a fast formation of the crystalline phase once the temperature of the expansion area 22 is reduced to below the freezing point of water. Advantageously, the cooling of the expansion area 22 can be decoupled in terms of time from the cooling of the remaining pressure vessel 10, as shown in FIG. 13B. A cooling device 200 as shown in the diagram with a cooling bath 210 is provided for cooling. The cooling device 200 comprises, for example, a vessel into which the cooling bath 210, e.g. of liquid nitrogen, is filled and which is connected with a cooling medium reservoir.

For cryopreservation of the biological sample 1 the cryopreservation device 100 is firstly immersed exclusively with the expansion area 22 into the cooling bath 210 (immersed depth D1) whilst the remaining pressure vessel is still above the cooling bath. In this phase, crystalline ice is formed in the expansion area 22 which expands such that the pressure increases in the internal space 12 of the pressure vessel 10. The duration of pressure generation with the expansion area 22 is selected, for example, in the range of milliseconds, seconds or minutes. Finally, the cryopreservation device 100 is completely immersed in the cooling bath 210 so that the desired cryopreservation temperature of −195.7° C., for example, is achieved. For this purpose, the cryopreservation device 100 is lowered to a second immersed depth D2.

The heating to recover the biological sample is conducted in reverse in accordance with FIG. 13C. To maintain the pressure in the pressure vessel 10, the internal space with the biological sample is firstly immersed in a heating bath 310 of a thawing device 300 (immersed depth D1) whilst the expansion area 22 is not yet cooled. After thawing of the biological sample 1 in the pressure vessel 10, the device is lowered to the immersed depth D2 so that the pressure in the pressure vessel 10 is also reduced.

FIG. 14A illustrates an embodiment of the inventive cryopreservation device 100 in which an inner vessel 13 is arranged in the internal space 12 of the pressure vessel 10. The inner vessel 13 is intended to receive the biological sample 1 and comprises a bubble-free filled tube of a flexible material. The inner vessel 13 is made, for example, of the same material as the vessel wall 12 of the pressure vessel 10. The provision of the inner vessel 13 has the advantage that the formation of the crystalline phase on the inner side of the vessel wall 12 is separated from the biological sample 1. Furthermore, different fluids can be provided in the internal space 12 outside the inner vessel 13 on the one hand and in the inner vessel 13 on the other. For example, pure water or an aqueous composition from water with a salt, glycerine and/or an alcohol can be provided outside the inner vessel 13. The aqueous composition has the advantage of reducing the freezing point so that the temperature at which the pressure in the pressure vessel 10 is to be generated can be determined below the freezing point of pure water.

The inner vessel 13 does not extend over the entire length of the internal space 12. This creates a relatively large space at the closed end 12.1 of the pressure vessel 10 in which no biological sample 1 is located and in which the crystalline phase of water can preferably be generated. This creates an expansion area within the pressure vessel 10 as part of the inventive actuating device which advantageously can have an effect on the cooling and on the heating of the cryopreservation device 100 (see in particular FIG. 14C).

The outer shape of the inner vessel 13 can be the same as the inner shape of the pressure vessel 10. By way of alternative to the cylindrical shape shown, other cross-sections of the outer or inner shapes can be provided such as quadratic, hexagonal, octagonal or all combinations thereof. In particular, different cross-section shapes of the outer and inner shapes can be provided.

FIG. 14B illustrates diagrammatically the cooling of the cryopreservation device 100 in a cooling device 200 which in this case contains two cooling baths 210 on top of one and other with liquid nitrogen and 220 with liquid propane. The use of liquid propane has the advantage that this wets the outer side of the pressure vessel 10 more easily and therefore accelerates cooling.

To heat the biological sample 1 in the pressure vessel 10 it is immersed in a heating bath 310 in accordance with FIG. 14C. The pressure vessel 10 can be oriented such that the entire length of the pressure vessel 10 is immersed simultaneously into the heating bath 310 (horizontal alignment). In this case, the temperature of the biological sample 1 is firstly increased and, as soon as the crystalline ice has melted, the pressure in the pressure vessel 10 is reduced. Alternatively, the closed end 12.1 with the expansion area can firstly be immersed in the water bath 310 so that the crystalline phase is firstly melted and the pressure in the pressure vessel 10 reduced and finally the remaining biological sample 1 heated (vertical alignment of the pressure vessel 10).

FIG. 15 illustrates diagrammatically that the use of the inventive cryopreservation device 100 is not restricted to the preservation of cell suspensions. Rather, the biological sample 1 can contain cell groups, cell aggregates, organs 4 of biological organisms or complete biological organisms 5 such as nematodes, worms or arthropods. For this purpose, the internal space 12 of the pressure vessel 10 has an inner diameter in the range of at least 5 mm, preferably at least 10 mm. The inner diameter of the internal space 11 is preferably smaller than 5 cm, in particular preferably less than 3 cm. As in FIG. 14, an inner vessel 13 is provided to receive the biological sample 1 which can be optionally subdivided into individual chambers.

The cryopreservation of the biological sample 1 is also conducted in the embodiment in accordance with FIG. 15 by the pressure vessel 10 being immersed in at least one liquid bath of the cooling device 200. In accordance with FIG. 15C, the pressure vessel 10 is first immersed with horizontal alignment in the liquid bath 220 with liquid propane and then in the liquid bath 210 with liquid nitrogen. By choosing the alignment of the pressure screw 21 relative to the liquid bath 220, 210, the time function of the pressure generation can be set relative to the time function of the cooling.

Heating is conducted in accordance with FIG. 15D in reverse order by firstly immersing the pressure vessel 10 in the heating bath 310 until the biological sample 1 has thawed. The cryopreservation device 100 is then completely lowered into the heating bath 310.

FIGS. 16 and 17 show further embodiments of the cryopreservation device 100 which is provided with an expansion area 22. As in FIG. 13, the expansion area 22 comprises a duct which has branches in the illustrated variants. In accordance with FIG. 16A, the expansion area 22 has a T-shape with lateral arms. The expansion area 22 can first be immersed in the cooling bath 210 (immersed depth D1) so that the liquid inside freezes, hexagonal ice is formed and the pressure increased. Only then is the system lowered entirely (immersed depth D2) and completely deep frozen (FIG. 16B). The reverse process is applied for heating or the possibility is provided to immerse into the heating bath 310 in two layers (arms first into the warm phase or last as shown here) (FIG. 16C). According to the latter principle, the pressure is maintained by the existing ice up to thawing at values of around 200 MPa.

FIG. 17 shows a further embodiment of the expansion area 22 with branches which form a fanned arm system. A larger inner volume and a larger surface than provided, for example in FIG. 16, results so that the pressure can be increased more quickly.

FIG. 18 shows embodiments of the cryopreservation device 100 with a spherical pressure vessel 10 and one expansion area 22 comprising several hollow ducts protruding in different axes (directions). The pressure vessel 10 comprises a hollow sphere to receive the biological sample. The hollow sphere is made of stainless steel, for example, and has an inner diameter of 10 mm and an outer diameter of 12 mm. The ducts form stubs protruding from the hollow sphere. The number, geometrical dimensioning and alignment of the ducts can be selected as dependent on the specific conditions of use (see examples in FIG. 18A to 18C). The hollow sphere is filled through a closable opening (not shown) in the vessel wall or through one of the ducts, which in this case is adapted with a closing element. In these embodiments too, the geometric alignment of the pressure vessel 10 permits a setting as to when the pressure in the pressure vessel 10 is to increase and drop on immersion to the immersed depth D1 and D2 in a cooling bath 210 (FIG. 18D) or in a heating bath 310 (FIG. 18E).

FIGS. 19A and 19B show embodiments of the cryo-preservation device 100 in which the vessel wall 11 of the pressure vessel 10 have the shape of a curved tube with simple curvature (FIG. 19A) or multiple curvature (FIG. 19B). In both cases, the pressure vessel 10 is bent in one bending level so that the local temperature distribution on immersion in the cooling bath can be set relative to the cooling bath depending on the alignment of the pressure vessel 10 (see FIG. 19C, 19D). FIG. 19A shows by way of example variants of pressure setting elements comprising pressure screws 21 and an expansion area 22 which can be provided individually or in combination as shown. The pressure screws 21 are formed as described above with reference to FIG. 5. The expansion area 22 comprises a large number of ducts which are arranged in the bending level of the pressure vessel 10 and which each communicate with the internal space of pressure vessel 10. Pressure setting elements can also be provided in the variant shown in FIG. 19B. The multiple bending in accordance with FIG. 19B can be shaped as a wave with more than the three extremes shown.

The local distributions and time functions of the pressure generation and the temperature reduction in the cryopreservation device 100 depend on the alignment of the pressure vessel 10 on immersion in the cooling bath 210. FIG. 19C shows, for example, an immersion with the bending level vertically or parallel to the surface of the cooling bath 210. Pressure generation and temperature lowering with the vertical alignment is conducted firstly in the extremes of the multiple bending which protrude into the cooling bath. By contrast, with the parallel alignment, pressure generation and temperature reduction is conducted simultaneously in the entire pressure vessel 10. Contrary to the illustrations, immersion with other alignments into the liquids is possible, leading to an extended variability of the settings of the pressure and temperature-time functions.

The local distributions and time functions of the pressure reduction and of the temperature increase in the cryopreservation device 100 similarly depend on the alignment of the pressure vessel 10 on immersion into a heating bath 310 (FIG. 19D).

FIG. 20 shows an embodiment of the cryopreservation device 100 in which the vessel wall 11 of the pressure vessel 10 has the shape of a flat or disc-like cylinder. This embodiment of the invention has the advantage that on immersion in a cooling bath a relatively large area is cooled. Therefore, a thin layer of the crystalline phase is formed on the inside of the vessel wall 11 which is sufficient to generate the required pressure in the pressure vessel 10 and simultaneously leave a relatively large amount of space for the vitreous phase. The pressure vessel 10 in accordance with FIG. 20A has a diameter of, for example, 1 cm to 10 cm and a thickness of, for example, 1 mm to 1 cm. It is made, for example, of stainless steel, aluminium, gold or silver.

In accordance with FIG. 20B, the actuating device for the time and/or location dependent setting of the temperature and of the pressure in the pressure vessel 10 comprises a pressure clamp 23. The pressure clamp 23 comprises two clamp plates 24 which can be pivoted via a hinge 25 (axial hinge). The distance between the clamp plates 24 can be set using a clamp screw 26 (setting screw). The pressure clamp 23 is suitable to apply pressure to the pressure vessel 10 which is higher than atmospheric pressure before the cryopreservation device 100 is cooled by immersion in a cooling bath (see FIG. 24). Furthermore, the pressure clamp 23 advantageously permits a stabilisation of the pressure vessel 10.

FIGS. 20C to 20D show variants of the cylindrically shaped pressure vessel 10 which can be adapted with a reinforced circumference area (perspective section in FIG. 20C) and/or a vessel wall 11 which can be deformed elastically on both sides (section in FIG. 20D) or on one side (section in FIG. 20E). In the latter case, a stable floor wall 11.3 is provided on one side.

FIGS. 21 to 24 show further variants of the pressure clamp 23 in which the clamp plates 24 are adapted with holes 27 and with profiles 28 on the inner side facing the pressure vessel 10 in open state and in closed state (assembled state) of the pressure clamp 23. In closed state of the pressure clamp 23 mechanical pressure is exerted on the vessel wall 11 of the pressure vessel 10 via the clamp plates 24. FIG. 23 shows a top view of the pressure clamps 23 with one of the clamp plates 24, the hinge 25, the clamp screw 26 and the holes 27.

The holes 27 permit direct contact of a cooling liquid, e.g. of liquid nitrogen or propanol, or of a heating fluid, e.g. of water, with the pressure vessel 10 and therefore an acceleration of cooling or heating of the pressure vessel 10. The profiles 28 are provided to generate different pressures at the pressure vessel 10 locally. It is furthermore shown that the cylindrical pressure vessel 10 can be adapted with a bleed connector 30.

FIG. 25 shows a variant of the invention in which the cryopreservation device 100 is immersed only on one side into a liquid (cooling bath 210 or heating bath). In this case the pressure clamp 23 has holes 27 only on the lower clamp plate 24 facing the liquid. This is particularly advantageous if adherent cells grow on the lower side of the pressure vessel 10 or the cells sediment down to it.

FIG. 26 illustrates diagrammatically a variant of the invention in which the bleeding of the pressure vessel 10 and the mechanical pressure generation can be controlled with the clamp plates 24 with electrical drive elements 32, 33. This embodiment of the invention is particularly advantageous for an automation of cryopreservation.

In an altered embodiment of the cryopreservation device 100 shown in FIG. 27 in which the vessel wall 11 of the pressure vessel 10 has the shape of a cylinder, no clamp but rather a pressure screw 21 is provided as pressure setting element. Using the pressure screw 21 the pressure in the internal space 12 of the pressure vessel 10 can be set before or after the start of cooling or heating in the cooling or heating bath (not shown). Several inner vessels 13 are arranged in the internal space 12 which are filled gas-free with biological samples, e.g. cell suspensions and are arranged, for example, stacked on top of one another. The wall thickness of the inner vessel 13 can be extremely thin, e.g. thinner than 400 μm because they are not exposed to pressure or corresponding mechanical forces.

According to FIG. 27, a sensor device comprising a pressure sensor 16.1 is arranged in the internal space 12 of the pressure vessel 10. The pressure sensor 16.1 provides a sensor signal which can be used to control the pressure using the pressure screw 21, possibly with an electrical control element (e.g. piezo element, not shown). A predetermined outer pressure can be generated which acts on the inner vessel 13 in the internal space 12 during freezing and thawing.

An altered variant of the pressure vessel 10 with pressure screw 21 is shown in FIG. 28. In this case the inner vessel 13 is formed by a flexible bag which receives the biological sample, e.g. a cell suspension or blood sample. The inner vessel 13 comprises, for example, a blood bag as used for blood donation purposes.

FIG. 29 illustrates diagrammatically an enlarged section of the vessel wall 11 of a pressure vessel 10. The vessel wall 11 contains an optical unit 60 which is adapted for a visual, in particular microscopic, observation of the internal space 12 of the pressure vessel 10. It is possible, for example, for cells 2 to adherently grow on an optical lens 61. The optical lens 61 can be configured, for example, for a microscopic image of the cells 2.

FIG. 30 shows a further embodiment of the invention in which the cryopreservation device 100 comprises a tube arrangement with external pressure generation (hydrostatic, see arrow) via a T element. The tube arrangement is adapted with a pressure sensor 16.1 and a temperature sensor 16.2 using the sensor signals of which the pressure and the temperature-time functions can be regulated.

FIGS. 31 and 32 show embodiments of the cryopreservation device 100 in which the vessel wall 11 of the pressure vessel 10 has the shape of a sphere. The pressure vessel 10 is composed of two semi spheres 11.2 which are screwed together in the middle of the pressure vessel 10. The spheres have a diameter in the range of, for example, 5 mm to 10 cm. According to FIG. 31A, the pressure vessel 10 is adapted with a pressure screw 21 which is used to fill the pressure vessel 10, bleed the pressure vessel 10 and generate the pressure in the internal space of the pressure vessel 10. FIG. 31B illustrates additionally optional parts such as a sensor device 16 and a filling line 11.1 which is decoupled from the pressure vessel 10. In analogous application of the above-described procedures, FIGS. 31C and 31D illustrate the cooling and heating of the cryopreservation device 100.

The spherical pressure vessel 10 can be alternatively or additionally provided with a pressure setting element in the shape of an expansion area 22 (FIG. 32). The expansion area 22 comprises a cylinder connection which, as explained above, is configured to receive an expansion medium or to generate pressure during cooling. In analogous application of the above-described procedures, FIGS. 32C and 32D illustrate the cooling and heating of the cryopreservation device 100.

The inventive cryopreservation of biological samples comprising organs 4 or entire organisms 5 is illustrated diagrammatically in FIGS. 33 to 35. Particularly in these applications of the invention, the biological sample is preferably combined with the stabiliser substance. According to FIG. 33A, a cylindrical vessel is provided as pressure vessel 10 which, in the example shown, is dimensioned to receive a fish embryo. Two immersion variants for pressure-temperature control are shown in FIGS. 33C and 33D for the freezing of the cryopreservation device 100. A spherical vessel is shown as pressure vessel 10 in accordance with FIGS. 34A and 34B which is dimensioned to receive an organ 4 with an inner diameter of the pressure vessel 10 in the range of, for example, 10 cm to 30 cm. According to FIG. 34B, the organ 4 is arranged in an inner vessel 13. FIG. 35 shows a spherical vessel as pressure vessel 10 in which tissue, organisms 5 or organs in an inner vessel 13, for example a bag or a thin-walled vessel, are located so that the outer solution to the inner bag medium can be different (e.g. outside oil, inside nutrient with a stabiliser substance).

FIG. 36 shows an embodiment of the cryopreservation device 100 in which the pressure is formed not by the expansion of a freezing expansion medium but by a vaporous expansion medium, e.g. vaporising liquid nitrogen. The expansion area 22 which contains the liquid nitrogen is connected to the pressure vessel 10 via a pressure line 39. The vaporising liquid nitrogen is injected continuously or in portions into the pressure vessel 10 from the expansion area 22.

FIGS. 37 to 39 show embodiments of the cryopreservation device 100 which are configured by the provision of at least one substrate 14 in the internal space 12 of the pressure vessel 10 for the cryopreservation of adherent cells 2. According to the variants shown in FIGS. 37A to 37C, the substrate 14 is located in the form of a long extended strip (tongue) in a tubular pressure vessel 10 which is closed on one or both sides by way of a pressure screw 21 and/or with a radially protruding pressure screw 21. The cells 2 are in an adherent state on one or both sides on the substrate 14 which can be functionalised in a suitable manner (e.g. by coating with fibronectin, polylysine and/or growth factors). Furthermore, the internal space 12 of the pressure vessel 10 is filled with a preservation medium, possibly with the stabiliser substance.

FIG. 37C also shows a substance reservoir 17 which is arranged in the internal space 12 of the pressure vessel 10. Substance reservoir 17 is adapted for an introduction of at least one additional substance into the internal space 12. For example, substances can diffuse into the internal space 12 from the substance reservoir 17 during the freezing or thawing procedure. The substance reservoir 17 can have a reservoir wall, for example made of plastic, which can be destroyed under the effect of pressure in the internal space 12. Advantageously, this permits the additional substance to be released in the internal space 12 only once a specific pressure has been reached.

Instead of the stretched strip, complicated shapes of the substrate 14 can be used as shown, for example, in FIG. 38. In accordance with FIG. 38A, a substrate 14 is illustrated with a cross-like cross-section whereby it can be possible for the cells 2 to be arranged on all or only on some surfaces of the substrate 14. According to FIG. 38B, a substrate 14 is shown in the shape of a hollow cylinder in the inside of which the cells 2 are located. The hollow cylinder can be made, for example, from ceramics, plastic, cellulose or chitin with an extremely thin wall, in particular thinner than 250 μm. As for the use of the above described inner vessel, this advantageously enables the cells 2 in the inside to be subjected to other peripheral conditions and solutions as outside the substrate 14. According to further variants of the invention, the substrate 14 can comprise a body with a nanostructured or microstructured surface which is adapted optionally with growth or differentiation factors in gradients.

FIGS. 39A to 39E show by way of example that the substrate 14 can be configured as a separate component (shuttle) which can be pushed into the pressure vessel. The shuttle can, in particular, be formed with a closed or open shape.

FIGS. 39A and 40 furthermore illustrate a variant of an inventive actuating device to control the pressure in the pressure vessel 10. In this variant, a coiled section 31 is provided as pressure-setting element at at least one end of the tubular pressure vessel 10. By turning the coiled section 31 at the pressure vessel 10 filled gas free with the biological sample the pressure can be increased here before lowering the temperature.

FIG. 40A shows a tubular pressure vessel 10 with coiled sections 31. The biological sample 1 with the preservation medium 3 is located in the internal space 12 of the pressure vessel 10. The preservation medium 3 in which the cells 2 are located can contain the stabiliser substance in the form of a gradient (e.g. Ficoll or Percoll of different concentrations and/or molecular weights). According to FIG. 40B, additional partition walls 18 can be provided in the internal space 12 which prevent a rapid mixing of the biological sample 1 and, for example, a disintegration of the gradient and permit a separation of the cells into compartments. Optionally, as shown in FIG. 40C, additional substance reservoirs in the shape of hollow spheres 19 can be provided in the internal space which are suspended and set out in the preservation medium 3, can be destroyed at a certain pre-set temperature and release their content. For example, further anti-freeze and vitrification substances or media which support the cells during thawing can be released. It is illustrated in FIG. 40D that two non-miscible solutions can be arranged layered on top of one another in the pressure vessel. The cells 2 are located in the first solution. The pressure relationships on temperature change can be set via the freeze behaviour of the solutions.

FIG. 41 illustrates a tubular pressure vessel 10 which is closed on one side with a pressure screw 21 and on one side with a coiled section 31. The preservation medium 3 is located in the internal space in the form of layered solutions (e.g. Ficoll as gradient or top layers of different molecular weights, i.e. solutions with different density) as used in the density gradient centrifusion of cells. The cells 2 are added unilaterally to the gradient (FIG. 41A). The pressure vessel 10 is centrifuged in vertical form with the cells 2 so that different cell types collect on the border areas (FIG. 41B). The cryopreservation then takes place in this arrangement.

FIGS. 42A and 42B diagrammatically illustrate a further embodiment of the inventive cryopreservation device 100 and its use. The cryopreservation device 100 comprises a pressure vessel 10 closed on one side with a pressure screw 21 in whose internal space 12 the substrate 14 is arranged for the adherent reception of biological samples 2. The pressure screw 21 is furthermore adapted with a heat conducting element in the form of a cooling wire 38 which is integrated pressure-tight into the pressure screw 21 and extends from the internal space 12 to the environment of the pressure vessel 10. The cooling wire 38 is a metal wire, for example, made of silver. Outside the pressure vessel 10, a cooling wire 38 is concentrated into a compact bundle, e.g. in the form of a spiral, a ball, a cylinder or a sphere. On freezing the cooling wire 38 can first be immersed in the cooling bath 210, e.g. of liquid nitrogen whereby ice does not form on the vessel wall 11 on the inside of the tube but on the cooling wire 38 leading to an increase in pressure. Thereafter the system is completely immersed into the cooling bath 210.

The features of the invention disclosed in the above description, the drawings and the claims can be of importance individually and also in combination for the realisation of the invention in its different embodiments. 

1. Cryopreservation device which is adapted for cryopreservation of a biological sample, comprising: a pressure vessel with a vessel wall and an internal space which is adapted to receive the biological sample, wherein the pressure vessel is configured for cooling with a lowering of a temperature and an increase of a pressure in the pressure vessel and for the cryopreservation of the biological sample, the pressure vessel is provided with an actuating device which is connected with the vessel wall and comprises at least one pressure-setting element and at least one of at least one cooling element and at least one heat conducting element, and the actuating device is configured for a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel, wherein the pressure setting element comprises at least one of a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous expansion medium and communicates with the internal space, and a pressure clamp which acts from outside on the vessel wall.
 2. Cryopreservation device in accordance with claim 1 in which the pressure setting element comprises a coiled section which acts on one end of the pressure vessel.
 3. Cryopreservation device in accordance with claim 2 in which the expansion area comprises at least one hollow duct which protrudes from the pressure vessel.
 4. Cryopreservation device in accordance with claim 3 in which the at least one hollow duct has at least one of branches and protrusions to different directions from the pressure vessel.
 5. Cryopreservation device in accordance with claim 1 in which the cooling element comprises a cooling line which is arranged in the pressure vessel.
 6. Cryopreservation device in accordance with claim 1 in which the heat conducting element comprises at least one of a profile on an outer side of the pressure vessel, a profile on an inner side of the pressure vessel and heat conducting bodies in the inside of the pressure vessel.
 7. Cryopreservation device in accordance with claim 1 in which the vessel wall of the pressure vessel has a shape of a tube, a sphere or a flat cylinder.
 8. Cryopreservation device in accordance with claim 7 in which the vessel wall of the pressure vessel has the shape of a bent tube.
 9. Cryopreservation device in accordance with claim 1 in which the at least one of the following is provided in the internal space of the pressure vessel: an inner vessel adapted to receive the biological sample, a substrate which is adapted for adherent receipt of biological cells in the biological sample, a segmentation of the internal space into internal space sections, a sensor device with a least one pressure sensor and a temperature sensor, and a substance reservoir which is adapted to release a substance into the internal space.
 10. Cryopreservation device in accordance with claim 9 in which the substance reservoir comprises hollow spheres made of a pressure sensitive material which are arranged distributed throughout the internal space.
 11. Cryopreservation device in accordance with claim 1 in which the vessel wall includes an optical unit which is adapted for a visual observation of the internal space of the pressure vessel.
 12. Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, with the steps: provision of the biological sample in a pressure vessel with a vessel wall and an internal space, and cooling of the pressure vessel in a cooling device with lowering of a temperature and increasing of a pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase, the pressure vessel is provided with an actuating device which comprises at least one pressure setting element and at least one of at least one cooling element and at least one heat conducting element, and a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel using the actuating device, wherein the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall.
 13. Method in accordance with claim 12 in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps: increase in pressure in the pressure vessel with the pressure setting element, and then lowering of the temperature in the pressure vessel with the cooling device.
 14. Method in accordance with claim 13 in which the pressure in the pressure vessel is increased using a coiled section which acts on one end of the pressure vessel.
 15. Method in accordance with claim 12 in which the expansion area comprises at least one hollow duct which protrudes from the pressure vessel, and the pressure is increased in the pressure vessel by first the at least one hollow duct being immersed into a cooling bath of the cooling device followed by a remainder of the pressure vessel.
 16. Method in accordance with claim 12 in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps: lowering of the temperature in the pressure vessel with the cooling device, and subsequently increasing of the pressure in the pressure vessel with the pressure setting element.
 17. Method in accordance with claim 16 in which the cryopreservation medium includes a stabiliser substance which is suitable to stabilise the vitreous phase of a supercooled melt preferably up to a transition to a liquid state, on increasing the temperature of the biological sample.
 18. Method in accordance with claim 17 in which the stabiliser substance is at least one member selected from the group consisting of: long-chain uncharged polymers with a molecular weight greater than 500 g/mol, monosaccharides, ethylene glycol, di- and oligo saccharides polysaccharides, starch derivatives, sugar alcohols; water-soluble polymers colloids comprising nano particle dispersions, dendrimers, polycations, and polyanions.
 19. Method in accordance with claim 18 in which the stabiliser substance is at least one member selected from the group consisting of: saccharose epichlorhydrin copolymer, and silica gel coated with polyvinyl pyrrolidone.
 20. Method in accordance with claim 17, wherein the stabiliser substance in the cryopreservation medium has a concentration which is lower than 10%.
 21. Method in accordance with claim 17, wherein the stabiliser substance in the cryopreservation medium is arranged outside the biological cells.
 22. Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, comprising the steps: providing a cryopreservation device of claim 1, providing the biological sample in the pressure vessel of the cryopreservation device, and cooling of the pressure vessel with lowering of the temperature and increasing of the pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase, wherein the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall.
 23. Method in accordance with claim 12 with the following step: storage of the biological sample whilst maintaining the increased pressure.
 24. Method for the heating of a biological sample such as to maintain vitality, comprising biological cells and a cryopreservation medium in a frozen state which is arranged in a vitreous phase in a pressure vessel, wherein a temperature of the pressure vessel and of the biological sample is increased and simultaneously an increased pressure above atmospheric pressure is maintained in the pressure vessel.
 25. Method in accordance with claim 24 in which the increased pressure above the atmospheric pressure is maintained in the pressure vessel until the biological sample achieves a transition from the frozen or vitrified state to a liquid state.
 26. Method in accordance with claim 25 in which on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is reduced.
 27. Method in accordance with claim 26 in which on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is instantaneously reduced.
 28. Method in accordance with claim 26 in which the pressure in the pressure vessel is reduced by a contraction of the biological sample at the transition from the frozen or vitrified state to the liquid state.
 29. Method in accordance with claim 24 in which the increased pressure is at least one of at least 100 MPa and maximum 300 MPa.
 30. Method in accordance with claim 24 in which the cryopreservation medium contains a stabiliser substance which is suitable to stabilise the vitreous phase of the supercooled melt on increasing the temperature of the biological sample, preferably up to the transition.
 31. Method of using a stabiliser substance for the cryopreservation of biological samples, including the steps of increasing the temperature of a biological sample, comprising biological cells and a cryopreservation medium, and maintaining a vitreous phase of the biological sample by an effect of the stabiliser substance up to a transition to a liquid state. 